1. Field of the Invention
The present invention relates to methods and compositions for the growth of mammalian cells in culture, particularly the growth of hematopoietic cell cultures. The present invention also relates to a functioning in vitro human tissue system, which may serve as a model for hematopoiesis. The present invention further relates to a method for assaying the effect of a substance and/or physical condition on a human hematopoietic cell mass or the hematopoietic process. The present invention also relates to a method for controlling the lineage development in an in vitro human tissue system and cultures of cells in which the population of a particular cell type has been enhanced relative to the total cell population in the culture or depleted. In addition, the present invention relates to a method of bone marrow tranplantation, in which the tissue implanted into the donee has been cultured by the present method.
2. Discussion of the Background
All of the circulating blood cells in the normal adult, including erythrocytes, leukocytes, platelets and lymphocytes, originate as precursor cells within the bone marrow. These cells, in turn, derive from very immature cells, called progenitors, which are assayed by their development into contiguous colonies of mature blood cells in 1-3 week cultures in semisolid media such as methylcellulose or agar.
Progenitor cells themselves derive from a class of progenitor cells called stem cells. Stem cells have the capacity, upon division, for both self-renewal and differentiation into progenitors. Thus, dividing stem cells generate both additional primitive stem cells and somewhat more differentiated progenitor cells. In addition to the generation of blood cells, stem cells also may give rise to osteoblasts and osteoclasts, and perhaps cells of other tissues as well. This document describes methods and compositions which permit, for the first time, the successful in vitro culture of human hematopoietic stem cells, which results in their proliferation and differentiation into progenitor cells and more mature blood cells of a specific lineage.
Although there are recent reports of the isolation and purification of progenitor cells (see, e.g., U.S. Pat. No. 5,061,620 as representative), such methods do not permit the long-term culture of viable and dividing stem cells.
In the late 1970s the liquid culture system was developed for growing hematopoietic bone marrow in vitro. The cultures are of great potential value both for the analysis of normal and leukemic hematopoiesis and for the experimental manipulation of bone marrow, for, e.g., retroviral-mediated gene transfer. These cultures have allowed a detailed analysis of murine hematopoiesis and have resulted in a detailed understanding of the murine system. In addition, it has made possible retroviral gene transfer into cultured mouse bone marrow cells. This allowed tagging murine hematopoietic cells proving the existence of the multi-potent stem cell and of the study of the various genes in the process of leukemogenesis.
But while it has been possible to transfer retroviral genes into cultured mouse bone marrow cells, this has not yet been possible in cultured human bone marrow cells because, to date, human long-term bone marrow cultures have been limited both in their longevity and more importantly in their ability to maintain stem cell survival and their ability to produce progenitor cells over time.
Human liquid bone marrow cultures were initially found to have a limited hematopoietic potential, producing decreasing numbers of progenitor cells and mature blood cells, with cell production ceasing by 6 to 8 weeks. Subsequent modifications of the original system resulted only in modest improvements. A solution to this problem is of incalculable value in that it would permit, e.g., expanding human stem cells and progenitor cells for bone marrow transplantation and for protection from chemotherapy, selecting and manipulating such cells, i.e., for gene transfer, and producing mature human blood cells for transfusion therapy.
Studies of hematopoiesis and in vitro liquid marrow cultures have identified fibroblasts and endothelial cells within adhering layers as central cellular stromal elements. These cells both provide sites of attachment for developing hematopoietic cells and can be induced to secrete hematopoietic growth factors which stimulate progenitor cell proliferation and differentiation. These hematopoietic growth factors include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6).
Cultures of human bone marrow cells on such adherent layers in vitro however have been largely disappointing. Unlike related cultures from other species, such as mouse and tree shrew, human liquid marrow cultures fail to produce significant numbers of either nonadherent hematopoietic precursor cells or clonogenic progenitor cells for over 6 to 8 weeks. And although cultures lasting 3-5 months have been reported, no culture which stably produces progenitor cells from stem cells continuously for more than 4-6 weeks has been reported.
Moreover, nonadherent and progenitor cell production typically declined throughout even the short life of these cultures, so that it is not clear that stem cell survival or proliferation is supported at all by these cultures. Further, when studied in isolation, unstimulated bone marrow stromal cells secrete little if any detectable hematopoietic growth factors (HGFs).
The lack of stable progenitor cell and mature blood cell production in these cultures has led to the belief that they are unable to support continual stem cell renewal and expansion. It has therefore been presumed that the cultures either lack a critical stem cell stimulant(s) and/or contain a novel stem cell inhibitor(s). However, while explanations for failure to detect HGFs and uninduced stromal cell cultures have been suggested, the null hypothesis, which combines the failure to detect HGFs and the relative failure of human liquid marrow cultures, would be that the culture systems used in vitro do not provide the full range of hematopoietic supportive function of adherent bone marrow stromal cells in vivo.
Stem cell and progenitor cell expansion for bone marrow transplantation is a potential application of human long-term bone marrow cultures. Human autologous and allogeneic bone marrow transplantation are currently used as therapies for diseases such as leukemia, lymphoma and other life-threatening disorders. For these procedures however, a large amount of donor bone marrow must be removed to insure that there is enough cells for engraftment.
A culture providing stem cell and progenitor cell expansion would reduce the need for large bone marrow donation and would make possible obtaining a small marrow donation and then expanding the number of stem cells and progenitor cells in vitro before infusion into the recipient. Also, it is known that a small number of stem cells and progenitor cells circulate in the blood stream. If these stem cells and progenitor cells could be collected by phoresis and expanded, then it would be possible to obtain the required number of stem cells and progenitor cells for transplantation from peripheral blood and eliminate the need for bone marrow donation.
Bone marrow transplantation requires that approximately 1xc3x97108 to 2xc3x97108 bone marrow mononuclear cells per kilogram of patient weight be infused for engraftment. This requires the bone marrow donation of the same number of cells which is on the order of 70 ml of marrow for a 70 kg donor. While 70 ml is a small fraction of the donors marrow, it requires an intensive donation and significant loss of blood in the donation process. If stem cells and progenitor cells could be expanded ten-fold, the donation procedure would be greatly reduced and possibly involve only collection of stem cells and progenitor cells from peripheral blood and expansion of these stem cells and progenitor cells.
Progenitor cell expansion would also be useful as a supplemental treatment to chemotherapy and is another application for human long-term bone marrow cultures. The dilemma faced by the oncologist is that most chemotherapy agents used to destroy cancer act by killing all cells going through cell division. Bone marrow is one of the most prolific tissues in the body and is therefore often the organ that is initially damaged by chemotherapy drugs. The result is that blood cell production is rapidly destroyed during chemotherapy treatment and chemotherapy must be terminated to allow the hematopoietic system to replenish the blood cell supply before a patient is retreated with chemotherapy. It may take a month or more for the once quiescent stem cells to raise up the white blood cell count to acceptable levels to resume chemotherapy during which case the drop in blood cell count is repeated. Unfortunately, while blood cells are regenerating between chemotherapy treatments, the cancer has time to grow and possibly become more resistant to the chemotherapy drugs due to natural selection.
To shorten the time between chemotherapy treatments, large numbers of progenitor and immature blood cells could be given back to the patient. This would have the effect of greatly reducing the time the patient would have low blood cell counts, thereby allowing more rapid resumption of the chemotherapy treatment. The longer chemotherapy is given and the shorter the duration between treatments, the greater the odds of successfully killing the cancer.
The hematopoietic cells required for progenitor cell expansion may come from either bone marrow withdrawal or peripheral blood collection. Bone marrow harvests would result in collection of approximately 4xc3x97105 CFU-GM progenitor cells. Phoresis of 5 liters of peripheral blood would collect approximately 105 CFU-GM although this number could be increased to 106 CFU-GM by prior treatment of the donor with GM-CSF. Rapid recovery of a patient would require transfusion of approximately 1xc3x97108 to 5xc3x97108 CFU-GM. Therefore, expansion of bone marrow or peripheral blood to increase the number of CFU-GM would be of benefit to chemotherapy administration and cancer treatment.
Gene therapy is a rapidly growing field in medicine which is also of inestimable clinical potential. Gene therapy is, by definition, the insertion of genes into cells for the purpose of medicinal therapy. Research in gene therapy has been on-going for several years in several types of cells in vitro and in animal studies, and has recently entered the first human clinical trials. Gene therapy has many potential uses in treating disease and has been reviewed extensively. See, e.g., Boggs, Int. J. Cell Cloning. (1990) 8:80-96, Kohn et al, Cancer Invest. (1989) 7 (2):179-192, Lehn, Bone Marrow Transp. (1990) 5:287-293, and Verma, Scientific Amer. (1990) pp. 68-84.
The human hematopoietic system is an ideal choice for gene therapy in that hematopoietic stem cells are readily accessible for treatment (bone marrow or peripheral blood harvest), they are believed to posses unlimited self-renewal capabilities (inferring lifetime therapy), and upon reinfusion, can expand and repopulate the marrow. Unfortunately, achieving therapeutic levels of gene transfer into stem cells has yet to be accomplished in humans.
Several disorders of the hematopoietic system include thalassemia, sickle cell anemia, Falconi""s anemia, acquired immune deficiency syndrome (AIDS) and SCIDS (ADA, adenosine deaminase deficiency). These candidates include both diseases that are inherited such as hemoglobinopathies and virally caused diseases of the hematopoietic system such as AIDS.
A salient problem which remain to be addressed for successful human gene therapy is the ability to insert the desired therapeutic gene into the chosen cells in a quantity such that it will be beneficial to the patient. To date, no method for doing this is available.
There is therefore a considerable need for methods and compositions for the in vitro replication of human stem cells and for the optimization of human hematopoietic progenitor cell cultures, particularly in light of the great potential for stem cell expansion, progenitor cell expansion, and gene therapy offered by these systems. Unfortunately, to date, attempts to achieve such results have been disappointing.
An in vitro system that permitted the controlled production of specific lineages of blood cells from within a hematopoietic cell population would have many applications. Controlled production of red blood cells would permit the in vitro production of red blood cell units for clinical replacement (transfusion) therapy. As is well known, red cells transfused are used in the treatment of anemia following elective surgery, in cases of traumatic blood loss, and in the supportive care of, e.g., cancer patients. Similarly, controlled production of platelets would permit the in vitro production of platelets for platelet transfusion therapy, for example for cancer patients in whom thrombocytopenia is caused by chemotherapy. For both red cells and platelets, current volunteer donor pools are accompanied by the risk of infectious contamination, and availability of an adequate supply can be limited. Controlled in vitro production of specified lineage of mature blood cells circumvent these problems.
Controlled, selective depletion of a particular lineage of cells from within a hematopoietic cell population can similarly confer important advantages. For example, production of stem cells and myeloid cells while selectively depleting T-cells from a bone marrow cell population could be very important for the management of patients with human immunodeficiency virus (HIV) infection. Since the major reservoir of HIV is the pool of mature T-cells, selective irradication of the mature T-cells from a hematopoietic cell mass collected from a patient has considerable potential therapeutic benefit. If one could selectively remove all the mature T-cells from within an HIV infected bone marrow cell population while maintaining viable stem cells, the T-cell depleted bone marrow sample could then be used to xe2x80x9crescuexe2x80x9d the patient following hematolymphoid ablation and autologous bone marrow transplantation. Although there are reports of the isolation of progenitor cells (see, e.g., U.S. Pat. No. 5,061,620 as representative) such techniques are distinct from and should not be confused with the selective removal of T-cells from a hematopoietic tissue culture.
Another application of T-cell depletion is the prevention of graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation. GVHD is a major limiting factor in the success of allogeneic bone marrow transplantation. Depletion of T-cells from a stem/progenitor cell population prior to allogeneic transplant would directly reduce the incidence and severity of GVHD. This depletion in turn would greatly decrease the morbidity and mortality of allogeneic bone marrow transplantation. While there are currently many techniques available for depleting T-cells from bone marrow samples (see e.g. Antin, J. H. et al, Blood, vol. 78, pp. 2139-2149 (1991)) none of these techniques allow the concurrent expansion of the hematopoietic progenitor cell population. Thus all of the previously developed techniques result in a diminution in the ability of the bone marrow sample to successfully engraft, thereby resulting in an increased incidence of graft failure. There is accordingly a considerable need for a method for depleting T-cells from a human hematopoietic mononuclear cell population, while maintaining or increasing the hematopoietic progenitor cell pool within the hematopoietic cell sample.
In addition, if it were possible to establish a functioning in vitro human tissue system, one could then utilize such a system as a model to stude the effects of chemical substances and/or physical conditions on a human hematopoietic cell mass or the hematopoietic process itself. Thus, by culturing such a system in the presence of a selected chemical substance and/or physical condition and comparing the state of the culture (total cell population, relative abundance of particular cell type, concentration of cell products in growth medium, etc.) with that of an identical culture, cultured in the absence of the selected chemical substance and/or physical condition, it would be possible to ascertain the effect of the selected chemical substance and/or physical condition on the hematopoietic cell mass or the hematopoietic process. In this way, such a functioning in vitro human tissue system could be utilized in an assay to detect the effect of a chemical substance and/or physical condition on a human hematopoietic cell mass or the hematopoietic process itself.
A further application of selective cell removal is the purging of malignant cells from bone marrow cultures for autologous bone marrow transplantation of cancer patients in which the cancer has metastasized. If it were possible to maintain a viable and producted human hematopoietic in vitro culture under conditions, which would lead to the depletion and extinction of malignant cells, then one could utilize such a culture for an autologous bone marrow transplant after a bout of chemotherapy, without the consequence of reintroducing metastasized malignant cells to the patient via the bone marrow transplant.
Thus, there remains a need for a functioning in vitro human hematopoietic tissue system and methods and conditions for maintaining such a system.
Accordingly, it is an object of this invention to provide novel methods, including culture media conditions, for the in vitro replication of human stem cells.
It is another object of this invention to provide novel methods, including culture media conditions, for the optimization of human hematopoietic progenitor cell cultures.
It is another object of the present invention to provide a novel, functioning, in vitro hematopoietic tissue system which may serve as a model of hematopoiesis.
It is another object of the present invention to provide a novel, functioning, in vitro hematopoietic tissue system which is substantially free of T-cells and B-cells.
It is another object of the present invention to provide a novel, functioning, in vitro hematopoietic tissue system in which at least a portion of the stem cells present have been genetically transformed.
It is another object of this invention to provide a novel, functioning, in vitro bone marrow tissue system in which the lineages of blood cells, including stem cells, produced can be controlled.
It is another object of this invention to provide novel methods, including culture media conditions, for the optimization of human hematopoietic progenitor cell cultures and to control the lineage composition of the mature cells produced.
It is another object of the present invention to provide novel methods, including culture media conditions, for the optimization of human hematopoietic progenitor cell cultures and to control the linage composition of the mature cells produced, in which at least a portion of the mature cells are derived from stem cells which have been genetically transformed.
It is another object of this invention to provide novel methods, including culture media conditions, for the selective enhanced production of red blood cells.
It is another object of this invention to provide novel methods, including culture media conditions, for the depletion of T-cells and B-cells from a human hematopoietic cell population.
It is another object of this invention to provide novel methods, including culture media conditions, for removing malignant cells from human hematopoietic cell population.
It is another object of this invention to provide novel methods, including culture media conditions, for assaying the affect of a substance or substances on a human replicating hematopoietic cell population.
It is another object of the present invention to provide novel methods, including culture media conditions, for assaying the effect of a physical condition or conditions on a human replicating hematopoietic cell population.
It is another object of the present invention to provide novel methods, including culture media conditions, for assaying the effect of genetic transformation of stem cells on a human replicating hematopoietic cell population.
It is another object of the present invention to provide novel methods for performing bone marrow transplantation in which the bone marrow tissue implanted in a patient is obtained according to the present method.
It is another object of the present invention to provide novel methods for performing bone marrow transplantation in which the bone marrow tissue implanted in a patient has been depleted of T-cells and B-cells.
It is another object of the present invention to provide novel methods for performing bone marrow transplantation in which the bone marrow tissue implanted in a patient has been depleted of malignant cells.
It is another object of the present invention to provide novel methods for performing bone marrow transplantation in which the bone marrow tissue implanted in a patient has been enriched in the population of a particular cell type as compared to the total cell population.
It is another object of the present invention to provide novel methods for performing bone marrow transplantation in which the bone marrow tissue implanted in a patient comprises stem cells which have been genetically transformed.
The present invention is based on the inventors"" discovery of novel methods, including culture media conditions, which provide for in vitro human stem cell division and/or the optimization of human hematopoietic progenitor cell cultures. These methods rely on culturing human stem cells and/or human hematopoietic progenitor cells in a liquid culture medium which is replaced, preferably perfused, either continuously, periodically, or intermittently, at a rate of 1 milliliter (ml) of medium per ml of culture per about 24 to about 48 hour period, and removing metabolic products and replenishing depleted nutrients while maintaining the culture under physiologically acceptable conditions. In a particularly preferred embodiment of the present invention, the above medium replacement rate is used in conjunction with the addition of hematopoietic growth factors to the rapidly exchanged culture medium.
The inventors have discovered that the increased medium exchange rate used in accordance with the present invention, with the optional addition of hematopoietic growth factors to the rapidly exchanged culture medium, surprisingly (1) supports cultures in which human stem cells proliferate over extended periods of time of at least 5 months, (2) supports cultures in which human hematopoietic progenitor cells are produced by division and differentiation of human stem cells through extended culture periods of at least 5 months, (3) stimulates the increased metobolism of and growth factor, including GM-CSF, secretion from human stromal cells, including human bone marrow stromal cells, 4) provides for the depletion of T-cells and B-cells from a human hematopoietic mononuclear cell population, (5) provides a method for assaying the affect of a substance or substances or physical conditions on a human hematopoietic cell population, (6) provides for the depletion of malignant cells from a human hematopoietic cell population, and (7) supports cultures in which human stem cells continue to divide over long periods of time and, thus, may be genetically transformed with a suitable vector such as a retrovirus. The present invention provides, for the first time, human stem cell survival and proliferation in culture. In addition, the present invention provides a functioning human in vitro tissue system which may serve as a model for a human hematopoietic cell mass of the process of hematopoiesis.
The advantages of the present invention may be observed whenever the present invention is applied to any standard system for liquid human hematopoietic culture. By the use of the rapid medium exchange rates used in accordance with the present invention, with the optional addition of supplementary hemotopoietic growth factors to the culture, the inventors have suprisingly discovered that one is able to make standard systems for liquid human hematopoietic cultures, which comprises cultures performed in the presence or absence of animal sera or plasmas, including horse, calf, fetal calf, or human serum, perform in a qualitatively superior manner. Human liquid hematopoietic cultures which may be used in accordance with the invention can be performed at cell densities of from 104 to 5xc3x97108 cells per ml of culture, using standard known medium components such as, for example, IMDM, MEM, DMEM, RPMI 1640, Alpha Medium or McCoy""s Medium, which can use combinations of serum albumin, cholesterol and/or insulin, transferrin, lecithin, selenium and inorganic salts. As known, these cultures may be supplemented with corticosteriods, such as hydrocortisone at a concentration of 10xe2x88x924 to 10xe2x88x927 M, or other corticosteriods at equal potent dose such as cortisone, dexamethasone or Solu-Medrol(copyright) (Upjohn).
These cultures are typically carried out at a pH which is roughly physiologic, i.e. 6.9 to 7.6. The medium is kept at an oxygen concentration that corresponds to an oxygen-containing atmosphere which contains from 1 to 20 vol. percent oxygen, preferably 3 to 12 vol. percent oxygen. The preferred range of O2 concentration refers to the concentration of O2 near the cells, not necessarily at the point of O2 introduction which may be at the medium surface or through a membrane. Using these standard culture techniques, the cell mass used may be enriched, by any desired amount, such as by up to 103 fold or more, either for stem cell content or for hematopoietic progenitor cell content. Different known methods may be used to achieve this enrichment, corresponding either to a negative selection method or a positive selection method. For example, in accordance with the negative selection method, mature cells are removed using immunological techniques, e.g., labelling non-progenitor, non-stem cells with a panel of mouse anti-human monoclonal antibodies, then removing the mouse antibody-coated cells by adherence to rabbit-anti-mouse Ig-coated plastic dishes. See e.g., Emerson et al, J. Clin. Invest. (1985) 76:1286-1290. Via such procedures, stem cells and progenitor cells may be concentrated to any degree desired.
The present invention relies on a fundamental alteration of the conditions of liquid human bone marrow cultures under any of the above conditions; rapid replacement of the nutrient medium. Standard culture schedules call for medium and serum to be exchanged weekly, either as a single exchange performed weekly or a one-half medium and serum exchange performed twice weekly. In accordance with the present invention, the nutrient medium of the culture is replaced, preferably perfused, either continuously or periodically, at a rate of about 1 ml per ml or culture per about 24 to about 48 hour period, for cells cultured at a density of from 2xc3x97106 to 1xc3x97107 cells per ml. For cell densities of from 1xc3x97104 to 2xc3x9710 6 cells per ml the same medium exchange rate may be used. Thus, for cell densities of about 107 cells per ml, the present medium replacement rate may be expressed as 1 ml of medium per 107 cells per about 24 to about 48 hour period. For cell densities higher than 107 cells per ml, the medium exchange rate may be increased proportionality to achieve a constant medium and serum flux per cell per unit time. Replacement of the nutrient medium in accordance with the invention may be carried out in any manner which will achieve the result of replacing the medium, e.g., by removing an aliquot of spent culture medium and replacing it with a fresh aliquot. The flow of the aliquot being added may be by gravity, by pump, or by any other suitable means, such as syringe or pipette. The flow may be in any direction or multiplicity or directions, depending upon the configuration and packing of the culture. Preferably, the new medium is added to the culture in a manner such that it contacts the cell mass. Most preferably, it is added to the culture in a manner mimicking in vivo perfusion, i.e., it is perfused through at least part of the cell mass and up to the whole cell mass.
Another, optional but important, embodiment of the present invention, resides in the addition of the hematopoietic growth factors to the rapidly exchanged cultures. In a particularly preferred aspect of this embodiment, the cytokines IL-3 and GM-CSF are both added, together, to the medium at a rate of from 0.1 to 100 ng/ml/day, preferably about 0.5 to 10 ng/ml/day, most preferably 1 to 2 ng/ml/day. Epo may be added to the nutrient medium in an amount of from 0.001 to 10 U/ml/day, preferably 0.05 to 0.15 U/ml/day. Mast cell growth factor (MCF, c-kit ligand, Steel factor), may be added to the medium in an amount of from 1 to 100 ng/ml/day, preferably 10 to 50 ng/ml/day. IL-1 (xcex1 or xcex2) may also be added in an amount of from 10 to 100 units/ml per 3 to 5 day period. Additionally, IL-6, G-CSF, basic fibroblast growth factor, IL-7, IL-8, IL-9, IL-10, IL-11, PDGF, or EGF may be added, at a rate of from 1 to 100 ng/ml/day.
The metabolic product level in the medium is normally maintained within a particular range. Glucose concentration is usually maintained in the range of about 5 to 20 mM. Lactate concentration is generally maintained in the range of from about 1 to 3 mM. Ammonium concentration is usually maintained below about 2.4 mM. These concentrations can be monitored by either periodic off line or on line continuous measurements using known methods. See, e.g., Caldwell et al, J. Cell Physiol. (1991) 147:344-353. The cells which may be cultured in accordance with the present invention may be human peripheral blood mononuclear cells, human bone marrow cells, human fetal liver cells, human cord blood cells, and/or human spleen cells. Each of these cell masses contains human stem cells and human hematopoietic progenitor cells.
In a preferred embodiment of the invention, the cell culture may be enriched to augment the human stem cell content of the cell mass. Such enrichment may achieved as described above, and, when used in accordance with the invention, provides the first useful means for genetic therapy via gene transfer into human bone marrow stem cells. In this embodiment, a packing cell line infected with a retrovirus, or a supernatant obtained from such a packaging cell line culture, is added to human stem cells cultured in accordance with the invention to obtain transformed human bone marrow stem cells. Such genetic transformation of human stem cells may be carried out an described in U.S. Pat. application Ser. No. 07/740,590 filed Aug. 5, 1991, now U.S. Pat. No. 5,399,493, which is incorporated herein by reference. The present invention provides increased levels of stem cell and human hematopoietic progenitor cell replication, whereas, by contrast, prior cultures provided only for human hematopoietic progenitor cell replication at a decreasing rate (i.e., decaying cultures). The present culture system provides, for the first time, expansion of cells in culture, which is required for retroviral infection was carried out on decaying cultures provided no infection of earlier cells. The preset invention, particularly when it is practiced together with an enriched stem cell pool, and even more particularly when it is practiced still further with the use of hematopoietic growth factors, provides a very effective means for obtaining stem cell infection in vitro.
In accordance with the present invention one obtains cultures in which human hematopoietic progenitor cells are produced by division and differentiation from human stem cells throughout a culture period of at least five months. That is, one obtains a culture which supports stem cell survival and proliferation in culture.
Data obtained by the inventors indicates that medium perfusion rate is a very significant variable in determining the behavior of in vitro human bone marrow cultures. This data shows that when the medium exchange rate is increase from the traditional once per week Dexter rate to a daily medium exchange rate of 7 volumes per week, a significant effect on in vitro hematopoiesis is obtained. In experiments carried out by the inventors, all cultures displayed a significant loss of cells during the first 3 to 4 weeks. Following this decay, the cultures stabilized and the effect of a medium perfusion rate became more pronounced.
A 3.5 per week medium exchange rate led to the most prolific cultures in the absence of added growth factors and also to cultures of greatest longevity in terms of progenitor cell production. Of particular note, during weeks 4 to 10, the biweekly number of nonadherent cells produced was actually stable or increasing.
Over the entire course of the cultures, the cumulative number of cells produced after week 3.5 was almost three-fold greater than that which is produced under the traditional Dexter culture protocol. Further, stable production of progenitor cells is maintained until week 18.
Bone marrow stomal cells may or may not be present in the cultures of the invention. In typical cultures, stromal cells are present in the cell culture in an amount of approximately 10xe2x88x923 to 10xe2x88x921 (stromal cells/total cells).
In another aspect of the invention, the inventors discovered that the cultures of the invention surprisingly provide increased metabolism and GM-CSF and IL-6 secretion from human bone marrow stromal cells. Whereas no GM-CSF is detected in human bone marrow stromal cells supernatant, rapid medium exchange in accordance with the invention stimulates human bone marrow stromal cells to secrete 300 femtograms/ml/day to 200 picograms/ml/day of GM-CSF. Secretion of IL-6 by human bone marrow stromal cells is also increased by rapid medium exchange in accordance with the invention from 1 to 2 ng/ml/day to 2 to 4 ng/ml/day. This increase is observed both when only the rapid medium exchange rate of the invention is used, and when the rapid exchange rate together with the addition of hematopoietic growth factors is used. On the basis of data obtained by the inventors, the effect of the rapid medium exchange rates of the invention on human stromal cell production of cytokines should be observed with human stromal cells in any complex tissue culture system.
Illustratively, the medium used in accordance with the invention may comprise three basic components. The first component is a media component comprised of IMDM, MEM, DMEM, RPMI 1640, Alpha Medium or McCoy""s Medium, or an equivalent known culture medium component. The second is a serum component which comprises at least horse serum or human serum and may optionally further comprise fetal calf serum, newborn calf serum, and/or calf serum. The third component is a corticosteriod, such as hydrocortisone, cortisone, dexamethasone, Solu-Medrol(copyright) (Upjohn), or a combination of these, preferably hydrocortisone. The serum component can be replaced in whole or in part with any standard serum replacement mixture.
The compositional make up of various media which can be used are set forth below.
The serum component may be present in the culture in an amount of at least 1% (v/v) to 50% (v/v). The preferred range will depend on whether or not serum is being used alone or is, at least in part, replaced by a serum replacement. When using no serum replacement, the serum concentration may be preferably in the neighborhood of 10 to 30% (v/v). The third component, corticosteriod, may be present in an amount of from 10xe2x88x92M to 10xe2x88x924 M, and is preferably present in an amount of from 5xc3x9710xe2x88x926 to 5xc3x9710xe2x88x925 M. Alternatively, the serum component can be replaced by any of several standard serum replacement mixtures which typically include insulin, albumin, and transferrin, lecithin, selenium or cholesterol. See, Migliaccio et al, Exp. Hematol. (1990) 18:1049-1055, Iscove et al, Exp. Cell Res. (1980) 126:121-126, and Dainiak et al, J. Clin. Invest. (1985) 76:1237-1242.
In addition to supporting the proliferation of human hematopoietic stem cells, the applications of these same conditions leads to the controlled production or depletion of specific lineage of blood cells. The inventors have discovered that when IL-3 and Epo, with or without GM-CSF, are used as described above one obtains lineage specific development of red blood cells. The inventors have also observed that T and B lymphocytes are lost from these cultures, during the same period of time in which the myeloid progenitor and cell mass is increasing. The inventors have also observed that leukemic cells are lost from these cultures over time.
The inventors also observed that with the cultures of the invention T and B lymphocytes are lost over time. As noted above, there are several T-cell-derived diseases and therapeutic concerns. For example, the autoimmune deficiency diseases (e.g., AIDS) result because of abnormal T-cell function caused by direct viral infection. Since this order results as a direct infection of the mature T-cell, and is not derived from defective hematopoietic stem or progenitor cells selective eradication of the mature T-cells has notable potential therapeutic benefit.
T-cell depletion has other applications as well. A limiting factor to the improved success of allogeneic bone marrow transplant is T-cell mediated. Depletion of T-cells from a stem/progenitor cell population prior to allogeneic transplant would enhance the reingraftment success by reducing the T-cell mediated graft versus host-rejection response.
The inventors have discovered that the present methods, including the present culture media conditions, which allow for the in vitro replication and differentiation of human stem and hematopoietic progenitor cells do not allow for maintenance of all hematopoietic cell classes. Although in the present methods and composition, human stem and hematopoietic progenitor cells are capable of in vitro replication and differentiation, human T-cells and B-cells, a major class of peripheral blood cells, do not proliferate or maintain viability in these in vitro culture conditions.
More particularly, T-cells require, among other factors, the growth factor interleukin-2 (IL-2) to remain viable. T-cells grown without such proper support die in approximately 3 to 4 days in a medium substantially free of IL-2. As a result, over a period of time when the viability and proliferative capacity of human stem and hematopoietic progenitor cells are maintained in accordance with the invention, the human T-cells contained in the human hematopoietic mononuclear cell population die, if the medium is substantially free of IL-2.
In accordance with this embodiment of the invention, a mixed human hematopoietic mononuclear cell fraction can be effectively depleted of T-cells and B-cells, under conditions which allow for expansion of stem and progenitor cells.
This method of selective T-cell depletion of human hematopoietic cell populations has notable therapeutic value as noted above. These include the following two applications:
1. Supportive treatment alone, or when used as adjuvant therapy, for curative treatment of AIDS and related T-cell diseases resulting from the dysfunction of the mature T-cell because of viral infection or other T-cell-specific functional disruption. The present culture conditions can be used to deplete disease-causing T-cells, while allowing for the survival, with or without their expansion, of human hematopoietic stem and/or progenitor cells. The T-cell-depleted hematopoietic cell population can then be used for reingraftment of patient bone marrow. The reestablished marrow will then produce anew, normal T-cell population. In its application to AIDS therapy, this procedure itself would not serve to necessarily eradicate the HIV from the patient, and reinfection of the newly developed T-cell in vivo is likely. Accordingly, this therapy would be considered as supportive, but, if used with other virus-eradication procedures, this procedure is operative for curative treatment protocols as well.
2. Allogeneic bone marrow transplant whereby a human hemotopoietic stem/progenitor cell population is depleted of viable T-cells and then used to reestablish the hematopoietic system in a recipient individual. The depletion of the T-cell population will increase the prospect of successful reingraftment by decreasing the graft versus host rejection process.
The present method of T-cell and B-cell depletion should be contrasted with methods for isolating and purifying progenitor cells (see, e.g., U.S. Pat. No. 5,061,620 as representative). The reported methods do not provide a method for the long term culture of viable and replicating stem cells, while the present method affords just such a result.
It should be understood that the present method for controlling the lineage development in a human hematopoietic tissue system may be practiced in conjunction with genetic transformation of at least a portion of the stem cells in the hematopoietic tissue system.
In another embodiment, the present invention provides a method for assaying the effect of a substance or substances and/or physical condition on a hematopoietic cell culture or the process of hematopoiesis. In accordance with this embodiment, one may add to a cell culture, carried out in accordance with the invention, at least one substance suspected of having an affect, which may be either beneficial or detrimental, on the cell culture. One may then compare the cell culture state obtained in the absence of the substance being tested to the cell culture state obtained in the presence of the substance.
Compounds or substances which may be tested include those which are expected to exert some effect on the hematopoietic system. Such compounds include, for example, hematopoietic growth factors, drugs, hormones, etc.
It should be understood that the present assay also permits the determination of the effect of substances endogenously produced by the present hematopoietic system. The effect of an endogenously produced substance may be assayed by adding to the medium a compound which either reduces the effective concentration of the endogenously produced substance and/or inhibits the action of the endogenously produced substance. Examples of such compounds include monoclonal antibodies, which bind to and neutralize endogenously produced growth factors, and antagonists, which bind to and block growth factor receptors on the surface of cells.
The present assay may also be used to ascertain the effect of physical conditions on the hematopoietic system or hematopoietic process. Such conditions include, for example, temperature, pressure, light intensity, gravity, etc. The effect of temperature, pressure, and light intensity may be determined by varying these parameters using conventional techniques and apparatus, such as heaters, refrigerators, pressurized or reduced-pressure chambers, and light sources. The effect of gravity may be determined by, e.g., carrying out the present culture in a zero-gravity environment, such as the space shuttle. The present assay may also be used to determine the effect of the particular configuration of the cell culture chamber, such as the nature of the surface which supports the adherent cell population.
Of course, it should be understood that the present assay is not limited to determining the effect of a single chemical substance or physical condition but may be used to detect the effect of the combined action of any number of chemical substances and/or physical conditions.
The parameters which can be monitored in carrying out the present assay include the cell population profile of the hematopoietic cell culture, the total cell population, the relative population of any particular type of cell, the presence, absence, or concentration of any other substance in the medium being removed from the culture, the consumption of nutrients, the morphology of any or all of the particular cell types present in the culture, the lifetime and duration of the culture, and the kinetics of hematopoiesis.
Thus, the present invention provides a functioning in vitro human hematopoietic tissue system which may serve as a model for the study of the naturally occurring in vivo hematopoietic system and the process of hematopoiesis. Accordingly, the effect of any chemical substance and/or physical condition on the hematopoietic system or the process of hematopoiesis may be determined by the present assay. It should be recognized that the present assay exhibits a significant advantage in that the effects of (i) slow acting substances and/or conditions or (ii) substances and/or conditions for which the effect exhibits a lag phase may be readily determined, because the present system provide a long term functioning hematopoietic tissue system.
It should also be understood that the present assay may be carried out when at least a portion of the stem cells in the human hematopoietic tissue system have been genetically transformed. Such genetic transformation may be used to introduce genetic markers useful for the subsequent identification of cells derived from the transformed stem cells. In addition, such genetic transformation may also serve as a method for introducing into the medium the chemical substance to be studied. Thus, when stem cells are transformed with a gene encoding for a particular substance and the appropriate regulatory sequences, the production of the substance by either the stem cell or a cell derived therefrom will provide a constant source of the substance.
In another embodiment, the present invention provides an improved method of bone marrow transplantation. Thus, by culturing bone marrow tissue according to the present method may of the drawbacks attendant to conventional bone marrow transplants may be avoided.
Thus, as noted above, the present method of bone marrow transplantation may be advantageously applied in situations in which the bone marrow tissue has been previously removed from a patient, before the patient is subjected to either chemotherapy or radiation therapy for the treatment of cancer, and is then implanted in the same patient after completion of the session of therapy. Since the present methods permit the expansion of the hematopoietic culture, a smaller quantity of tissue may removed form the patient prior to therapy. In addition, since culture of the bone marrow tissue according to the present method results in the depletion and extinction of malignant cells, implantation of the tissue cultured according to the present method poses a reduced risk of reintroducing malignant cells which may have metastasized into the bone marrow.
In the setting of allogeneic bone marrow transplants, the present method also exhibits distinct advantages. Thus, because culturing according to the present method, using medium substantially free of IL-2, results in a bone marrow culture substantially free of T-cells and B-cells and since such cells are principally involved in graft versus host disease, implantation of allogeneic bone marrow tissue cultured according to the present method poses a reduced risk of graft versus host disease.
It should be stressed that these advantages are uniquely afforded by the present invention. Thus, it is the ability of the present culture techniques to maintain and/or expand a viable and functioning in vitro hematopoietic tissue system for a time sufficient to effect the depletion of malignant cells and/or T-cells and B-cells that enables the implantation of bone marrow tissue substantially free of malignant cells and/or T-cells and B-cells. Culturing bone marrow tissue, for a time sufficient to effect such depletions, by conventional techniques would not result in a functioning in vitro hematopoietic tissue system containing viable stem cells.
The present method of bone marrow transplantation may be carried out as follows: removing a tissue sample from a donor; culturing said tissue sample according to the present method; and implanting said cultured tissue in a donee. As noted above the donor and donee may be the same or different.
The tissue sample may be obtained from the donor according to conventional methods using conventional apparatus. Apparatus for and methods of bone marrow transplantation are disclosed in U.S. Pat. Nos. 4,481,946 and 4,486,188, which are incorporated herein by reference.
After the tissue sample has been obtained, it is then cultured according to the present method, for a time sufficient to achieve the desired expansion and/or cell depletion. The cultured tissue may then be implanted in the donee, again, according to conventional techniques.
It should be understood that the present method of bone marrow transplantation may be used advantageously in conjunction with genetic transformation of at least a portion of the stem cells in the tissue to be implanted in the donee. The advantages of stem cell transformation in gene therapy are discussed above. Thus, the present method of bone marrow transplantation, when used in conjunction with genetic transformation of stem cells in the implanted tissue, represents an improved method of gene therapy.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.